Method of storing tetrazolium compound, stabilizer for use therein, and tetrazolium compound reagent solution stored by the method

ABSTRACT

A method for storing a tetrazolium compound stably is provided. The tetrazolium compound is stored in the presence of sodium azide. The tetrazolium compound (A) and the sodium azide (B) are present at a ratio (A:B) in the range from 1:0.02 to 1:6.2. Furthermore, when the tetrazolium compound is stored as a solution, the concentration of the sodium azide is in the range from 0.08 to 3.2 mmol/L and the concentration of the tetrazolium compound is in the range from 0.5 to 8 mmol/L. As the tetrazolium compound, it is preferable to use 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium salt.

TECHNICAL FIELD

[0001] The present invention relates to a method for storing atetrazolium compound stably, a stabilizer used in the method, and atetrazolium compound reagent solution using the method.

BACKGROUND ART

[0002] Tetrazolium compounds generally are used as a redox dye (acolor-developing substrate), a reducing agent, or the like. To this end,a liquid reagent prepared by dissolving a tetrazolium compound in waterusually is used.

[0003] However, in the case where the pH of the tetrazolium compoundsolution is close to neutral, the tetrazolium compound exhibits lowstability. Thus, when the tetrazolium compound solution is stored for along time, there arises a problem in that the solution may be coloreddue to spontaneous color development of the tetrazolium compound or thatthe solution may no longer act as a reducing agent, for example. On thisaccount, when a tetrazolium compound solution needs to be used, there isno other way but to prepare a tetrazolium compound solution for each useor to use a tetrazolium compound solution stored as an acid solutionbecause the tetrazolium compound is stable even in the form of asolution under acidic conditions.

DISCLOSURE OF INVENTION

[0004] However, in the case where a tetrazolium compound is used in anenzyme reaction system, it is necessary to set various conditionsaccording to the reaction. In particular, enzymes have their own optimumpH and pH stability, and these optimum pH and pH stability are notnecessarily acidic. In fact, the optimum pH and pH stability of manyenzymes are alkaline, and in particular, there are a large number ofenzymes whose optimum pH and pH stability are close to neutral. Thus, inthe case where an enzyme reaction is caused at a pH close to neutral, ifan acid solution containing a tetrazolium compound is to be used, it isnecessary to adjust the pH of the reaction system when using thesolution. This makes the operation complicated.

[0005] Therefore, it is an object of the present invention to provide amethod for storing a tetrazolium compound stably not only under acidicconditions but also under other pH conditions.

[0006] In order to achieve the above object, the present inventionprovides a method for storing a tetrazolium compound stably, including:storing the tetrazolium compound in the presence of sodium azide. In thepresence of the sodium azide, the tetrazolium compound can be stored ina stabilized condition where, for example, the spontaneous colordevelopment thereof is suppressed and the function thereof is maintainednot only under acidic conditions but also under other pH conditions.

[0007] In the present invention, the tetrazolium compound may be storedin the form of a solution or in the dry state. In the case where thetetrazolium compound is stored in the dry state, the tetrazoliumcompound may be stored, for example, by adding sodium azide to asolution containing the tetrazolium compound and then drying thismixture as it is. Alternatively, filter paper or the like may beimpregnated with this mixture and then dried.

[0008] The sodium azide generally is used as an antiseptic. However, inthe present invention, the sodium azide is not added to produce anantiseptic effect but to store the tetrazolium compound in a stabilizedcondition where the spontaneous color development thereof is suppressedand the function thereof is maintained. It is the inventors of thepresent invention who discovered that sodium azide can stabilize atetrazolium compound.

[0009] In the method according to the present invention, it ispreferable that the tetrazolium compound (A) and the sodium azide (B)are present at a ratio (molar ratio A:B) in a range from 1:0.02 to1:6.2.

[0010] In the method according to the present invention, when thetetrazolium compound and the sodium azide are present in a solution sothat the tetrazolium compound is stored stably, it is preferable that aconcentration of the sodium azide is in a range from 0.08 to 3.2 mmol/L,more preferably 0.08 to 0.8 mmol/L. This is because the tetrazoliumcompound can be stored even more stably when the concentration of thesodium azide is in the above-described range. On the other hand, whenthe sodium azide is used as an antiseptic as described above, theconcentration of the sodium azide needs to be about 0.05 to 0.2 wt %(7.7 to 31 mol/L) in order to produce an antiseptic effect. However, inthe present invention, the sodium azide exhibits a particularlyexcellent effect of stabilizing the tetrazolium compound when theconcentration thereof is in the range from 0.08 to 3.2 mmol/L. Withinthis range, the sodium azide exhibits substantially no antisepticeffect. That is, it is considered that the stabilization of thetetrazolium compound is not achieved by the antiseptic effect of thesodium azide, and it can be said that the antiseptic effect and theeffect of stabilizing the tetrazolium compound are completely differentfrom each other.

[0011] On the other hand, in the method according to the presentinvention, it is preferable that a concentration of the tetrazoliumcompound is in a range from 0.5 to 8 mmol/L.

[0012] Furthermore, in the method according to the present invention, itis preferable that the sodium azide is added to the solution so that itsconcentration falls within the range from 0.02 to 6.2 mmol/L per 1mmol/L of the tetrazolium compound.

[0013] In the method according to the present invention, a pH of thesolution is not particularly limited, but is, for example, in the rangefrom 5.0 to 7.5, preferably 5.0 to 7.0, and more preferably 5.5 to 6.5.

[0014] In the method according to the present invention, it ispreferable that the tetrazolium compound is2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt.

[0015] Next, a stabilizer according to the present invention is astabilizer for storing a tetrazolium compound stably, which includessodium azide. The reason for this is that the sodium azide can stabilizethe tetrazolium compound as described above.

[0016] Next, a tetrazolium compound reagent according to the presentinvention is a reagent solution including an aqueous solvent and atetrazolium compound dissolved in the aqueous solvent, and sodium azidefurther is dissolved in the aqueous solvent. In such a reagent,spontaneous color development of the tetrazolium compound or loss of thefunction of the tetrazolium compound can be suppressed even if thereagent is in the form of a solution. Therefore, the necessity ofpreparing a reagent solution for each use is eliminated so thatoperations using a tetrazolium compound, such as various measurementreactions, can be carried out simply.

[0017] Furthermore, a dry reagent according to the present invention isa tetrazolium compound-containing reagent obtained by drying an aqueoussolvent in which the tetrazolium compound and sodium azide aredissolved. The aqueous solvent in which the tetrazolium compound andsodium azide are dissolved may be dried as it is. Alternatively, filterpaper or the like may be impregnated with the aqueous solvent and thendried.

BRIEF DESCRIPTION OF DRAWINGS

[0018]FIG. 1 is a graph showing the change in absorbance correspondingto an amount of glycated hemoglobin with time in a method for storing atetrazolium compound stably according to one example of the presentinvention.

[0019]FIG. 2 is a graph showing the change in absorbance correspondingto an amount of glycated hemoglobin with time in the same example as inFIG. 1.

[0020]FIGS. 3A and 3B are graphs showing the change in absorbance in amethod for storing a tetrazolium compound stably according to anotherexample of the present invention, wherein FIG. 3A shows the result withrespect to a sample having a WST-3 concentration of 0.5 mmol/L and asodium azide concentration of 0.05 g/L and FIG. 3B shows the result withrespect to a sample having a WST-3 concentration of 2.0 mmol/L and asodium azide concentration of 0.1 g/L.

BEST MODE FOR CARRYING OUT THE INVENTION

[0021] The tetrazolium compound to be used in a method for storing atetrazolium compound stably according to the present inventionpreferably contains ring substituents at least at two positions on itstetrazole ring, more preferably at three positions on its tetrazolering, for example.

[0022] In the case where the tetrazolium compound contains ringsubstituents at least at two positions on its tetrazole ring asdescribed above, it is preferable that the ring substituents are at the2-position and 3-position on the tetrazole ring. Further, in the casewhere the tetrazolium compound contains ring substituents at threepositions on its tetrazole ring, it is preferable that the ringsubstituents are at the 2-position, 3-position, and 5-position on thetetrazole ring.

[0023] Further, it is preferable that at least two ring substituents ofthe tetrazolium compound have a benzene ring structure. Other than thebenzene ring structure, the ring substituents may have a resonancestructure with S or O being contained in the ring skeleton, for example.Examples of the ring substituents with such a resonance structureinclude a thienyl group, thiazoyl group, and the like.

[0024] Furthermore, it is preferable that the tetrazolium compoundcontains ring substituents at least at three positions on its tetrazolering and at least two of the ring substituents have a benzene ringstructure.

[0025] Still further, it is preferable that at least one ringsubstituent contains a functional group, and a larger number offunctional groups are more preferable.

[0026] As the functional group, an electron-withdrawing functional grouppreferably is used. For example, a halogen group, ether group, estergroup, carboxy group, acyl group, nitroso group, nitro group, hydroxygroup, sulfo group, and the like can be used. Other than these,characteristic groups containing oxygen such as a hydroperoxy group, oxygroup, epoxy group, epidioxy group, oxo group, and the like; andcharacteristic groups containing sulfur such as a mercapto group,alkylthio group, methylthiomethyl group, thioxo group, sulfino group,benzenesulfonyl group, phenylsulfonyl group, p-toluenesulfonyl group,p-tolylsulfonyl group, tosyl group, sulfamoyl group, isothiocyanategroup, and the like also can be used, for example. Among theseelectron-withdrawing functional groups, a nitro group, sulfo group,halogen group, carboxy group, hydroxy group, methoxy group, ethoxy groupare preferable. Further, in addition to the above-mentionedelectron-withdrawing functional groups, unsaturated hydrocarbon groupssuch as a phenyl group (C₆H₅—), styryl group (C₆H₅CH ═CH—), and the likealso can be used, for example. It is to be noted that the functionalgroups may have been ionized by dissociation.

[0027] Still further, it is preferable that the tetrazolium compoundcontains benzene rings at the 2-position and 3-position on its tetrazolering and at least one of the benzene rings contains at least onefunctional group selected from the group consisting of a halogen group,carboxy group, nitro group, hydroxy group, sulfo group, methoxy group,and ethoxy group. It is to be noted here that both the benzene rings maycontain the functional group. Further, the functional group may becontained at any positions (ortho-, meta-, para-) on the benzene ring.Furthermore, the number of the functional group is not specificallylimited, and the benzene ring may have either the same or differentfunctional groups.

[0028] Examples of the tetrazolium compound containing ring substituentshaving a benzene ring structure at the 2-position, 3-position, and5-position on its tetrazole ring include:

[0029]2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt;

[0030]2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt;

[0031]2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt;

[0032] 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium salt;

[0033] 3,3′-(1,1′-biphenyl-4,4′-diyl)-bis(2, 5-diphenyl)-2H-tetrazoliumsalt;

[0034] 3,3′-[3,3′-dimethoxy-(1,1′-biphenyl)-4,4′-diyl]-bis[2-(4-nitrophenyl)-5-phenyl-2 H-tetrazolium salt];

[0035] 2,3-diphenyl-5-(4-chlorophenyl)tetrazolium salt;

[0036] 2,5-diphenyl-3-(p-diphenyl)tetrazolium salt;

[0037] 2,3-diphenyl-5-(p-diphenyl)tetrazolium salt;

[0038] 2,5-diphenyl-3-(4-styrylphenyl)tetrazolium salt;

[0039] 2,5-diphenyl-3-(m-tolyl)tetrazolium salt; and

[0040] 2,5-diphenyl-3-(p-tolyl)tetrazolium salt.

[0041] The tetrazolium compound is not limited to those described above.In addition to the above-mentioned tetrazolium compounds, a tetrazoliumcompound containing ring substituents having a benzene ring structure attwo positions and one ring substituent having a structure other than thebenzene ring structure at one position on its tetrazole ring also may beused. Examples of such a tetrazolium compound include:

[0042] 2,3-diphenyl-5-(2-thienyl)tetrazolium salt;

[0043] 2-benzothiazoyl-3-(4-carboxy-2-methoxyphenyl)-5-[4-(2-sulfoethylcarbamoyl)phenyl]-2H-tetrazolium salt;

[0044] 2,2′-dibenzothiazoyl-5,5′-bis[4-di(2-sulfoethyl)carbamoylphenyl]-3,3′-(3,3′-dimethoxy-4,4′-biphenylene)ditetrazoliumsalt; and

[0045] 3-(4,5-dimethyl-2-thiazoyl)-2, 5-diphenyl-2H-tetrazolium salt.

[0046] Further, a tetrazolium compound containing ring substituentshaving a benzene ring structure at two positions and one substituent nothaving a ring structure at one position on its tetrazole ring also canbe used. Examples of such a tetrazolium compound include:

[0047] 2,3-diphenyl-5-cyano tetrazolium salt;

[0048] 2,3-diphenyl-5-carboxy tetrazolium salt;

[0049] 2,3-diphenyl-5-methyltetrazolium salt; and

[0050] 2,3-diphenyl-5-ethyl tetrazolium salt.

[0051] Among the above-mentioned tetrazolium compounds, the tetrazoliumcompounds containing three ring substituents are preferable as describedabove. Among these, the tetrazolium compounds containing three ringsubstituents having a benzene ring structure and a large number ofelectron-withdrawing functional groups is more preferable, and2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt is most preferable. It is to be noted here that the above-mentionedtetrazolium compounds may be a salt or may have been ionized, forexample.

[0052] The storage of a tetrazolium compound according to the presentinvention may be carried out, for example, by dissolving a tetrazoliumcompound and sodium azide as a stabilizer in an aqueous solvent andstoring the thus-obtained tetrazolium compound aqueous solution. Theconcentration of the tetrazolium compound in the aqueous solution is notparticularly limited, but is, for example, in the range from 0.5 to 8mmol/L as described above, due to the solubility in water of thetetrazolium compound or the like.

[0053] On the other hand, the concentration of the sodium azide is, forexample, in the range from 0.08 to 3.2 mmol/L, more preferably 0.08 to0.8 mmol/L, as described above.

[0054] Furthermore, the sodium azide preferably is added to the solutionso that its concentration falls within the range from 0.02 to 6.2 mmol/Lper 1 mmol/L of the tetrazolium compound, for example.

[0055] As the aqueous solvent, water, various buffers, and the like canbe used, for example. As the buffers, a phosphate buffer, Good's buffers(MES, MOPSO, MOPS, DIPSO, TES, POPSO, and HEPES), and the like can beused. Among these, MES and MOPS are preferable, and MES is morepreferable. The pH of the buffer is, for example, in the range from 5.0to 7.5, preferably 5.0 to 7.0, and more preferably 5.5 to 6.5.

[0056] In the presence of sodium azide, the tetrazolium compound can bestored stably without adjusting the pH of the aqueous solution so as tobe acidic, as required conventionally. Thus, the pH of the aqueoussolution is not particularly limited, but is, for example, in the rangefrom 5.0 to 7.5, preferably 5.0 to 7.0, and more preferably 5.5 to 6.5.

[0057] The storage temperature of the aqueous solution containing thetetrazolium compound and the sodium azide is not particularly limited,but preferably is in the range from 4° C. to 60° C.

[0058] In the case where the aqueous solution is stored at 4° C., theaqueous solution can be stored for, for example, at least 90 days whilesuppressing spontaneous color development of the tetrazolium compoundand maintaining the function of the tetrazolium compound.

[0059] The tetrazolium compound stored in the above-described manner isuseful as a liquid tetrazolium compound reagent because spontaneouscolor development of the tetrazolium compound is suppressed and thefunction of the tetrazolium compound is maintained as described aboveeven if the tetrazolium compound is stored as a solution for a longtime. The application of the tetrazolium compound reagent is notparticularly limited. For example, the tetrazolium compound reagent maybe used as a color-developing substrate, a reducing agent, or the likeas described above.

[0060] Furthermore, by drying the aqueous solution as it is or dryingfilter paper or the like impregnated with the aqueous solution asdescribed above, the tetrazolium compound may be used as a dry reagent.

EXAMPLES Example 1 and Comparative Example 1

[0061] In Example 1, a tetrazolium compound was stored as an aqueoussolution in the presence of sodium azide, and the change in color in theaqueous solution was examined. As the tetrazolium compound, a productnamed “WST-3” (manufactured by Dojindo Laboratories, hereinafter thesame) was used.

[0062] Samples having the following compositions were prepared by addingsodium azide so that the samples contained the sodium azide atpredetermined concentrations (0.01, 0.03, 0.05, 0.07, 0.10, 0.20 g/L),respectively. These samples were stored at 4° C. for 8 days. After thestorage, the absorbance of these samples at the wavelength of 450 nm wasmeasured using a spectrophotometer (product name “Lambda 20”,manufactured by PerkinElmer, Inc., hereinafter the same). The resultsare shown in Table 1 below. The sample containing no sodium azide (0g/L) was regarded as Comparative Example 1. (Composition of Samples)PIPES buffer (pH 7.5)   5 mmol/L Tetrazolium compound 0.5 mmol/L Sodiumazide predetermined concentration

[0063] TABLE 1 Sodium azide (g/L) 0 0.01 0.03 0.05 0.07 0.10 0.20Absorbance 0.352 0.207 0.181 0.202 0.192 0.196 0.264

[0064] As shown in Table 1, in the presence of the sodium azide, theWST-3 could be stored stably with the color development of the WST-3being suppressed even though the WST-3 was stored as an aqueoussolution. Besides, even though the pH of the samples was close toneutral and not acidic as required conventionally, the WST-3 could bestored sufficiently stably. Furthermore, it can be said from the resultsthat the amount of the sodium azide to be added preferably is in therange from 0.01 to 0.2 g/L per 0.5 mmol/L of the WST-3.

Example 2, Example 3, and Comparative Example 2

[0065] In these examples, a tetrazolium compound and a metalloproteinasewere stored as an aqueous solution in the presence of sodium azide, andwhether or not the aqueous solution was colored and whether or not thefunction of the tetrazolium compound was maintained were examined.

[0066] (Storage Procedure)

[0067] Enzyme reagents containing a tetrazolium compound were preparedso as to have the following compositions. These reagents were stored atpredetermined temperatures (4° C. and 25° C.) and samples were takenfrom them at predetermined periods after the start of storage. Withrespect to the samples taken after the predetermined storage periods,whether or not the samples were colored was confirmed in the followingmanner. Further, measurement of glycated hemoglobin using each of thesesamples was carried out in the following manner. In Comparative Example2, an enzyme reagent was prepared in the same manner as that in Examples2 and 3 except that no sodium azide was added thereto, which was thenstored and subjected to the confirmation of coloring and the measurementof glycated hemoglobin in the same manner as that in Examples 2 and 3.As the metalloproteinase shown below, a product named“Metalloproteinase” (Toyobo Co., Ltd.) was used. (Composition ofTetrazolium Compound-Containing Enzyme Reagent) Ex. 1 Ex. 2 Com. Ex. 2Metalloproteinase  2.0 g/L ∘ ∘ ∘ WST-3  2.0 mmol/L ∘ ∘ ∘ MOPS buffer (pH6.5)  5.0 mmol/L ∘ — — MES buffer (pH 5.5)  5.0 mmol/L — ∘ ∘ NaN₃  0.05g/L ∘ ∘ — CaCl₂  1.0 mmol/L ∘ ∘ ∘ NaCl 300.0 mmol/L ∘ ∘ ∘

[0068] A. Method for Confirming Coloring

[0069] With respect to the enzyme reagent sample (storage period: 33days), the absorbance (at the wavelength of 450 nm) was measured usingthe above-described spectrophotometer. The results are shown in Table 2.TABLE 2 Change in absorbance (unit: absorbance) Ex. 2 Ex. 3 Com. Ex. 24° C. 25° C. 4° C. 25° C. 4° C. 25° C. 33 days after 0.191 0.284 0.0810.201 0.299 1.911 preparation

[0070] B. Measurement of Glycated Hemoglobin

[0071] In measurement of an analyte in a sample utilizing a redoxreaction, when the sample contains a reducing substance such as ascorbicacid or hemoglobin, the measurement may not be carried out accuratelybecause the reducing substance may reduce an oxide as the analyte, acolor-developing substrate may develop color, or color developed may bedisappeared, for example. In such cases, a tetrazolium compound iseffective because it has a function of eliminating the influence of thereducing substance, such as hindrance to a reaction or color developmenterror as described above, thereby improving the accuracy of themeasurement. Therefore, by carrying out the measurement of glycatedhemoglobin utilizing a redox reaction using a tetrazolium compoundstored as an aqueous solution in the presence of sodium azide, it ispossible to determine whether or not the tetrazolium compound stillmaintains its function after the storage.

[0072] In this measurement of glycated hemoglobin, the amount of theglycated hemoglobin is determined by degrading glycated hemoglobin witha protease; reacting a fructosyl amino acid oxidase (hereinafter,referred to as “FAOD”) with a glycated side chain group of an amino acidresidue in the degradation product so that hydrogen peroxide isgenerated; causing a redox reaction between the hydrogen peroxide and acolor-developing substrate; and then measuring the color development ofthe substrate. Specific procedures for this measurement will bedescribed in the following.

[0073] First, each measurement sample shown below was diluted 2-fold (byvolume), and 25 μL of this diluted solution was mixed with 60 μL of eachof the enzyme reagent samples taken after the storage for predeterminedperiods and 25 μL of a color-developing reagent shown below. Theobtained mixtures (110 μL) were allowed to react for 15 minutes at 37°C. Thereafter, the absorbance of the mixtures at the main wavelength of751 nm and the sub-wavelength of 805 nm was measured using a biochemicalautomatic analysis apparatus (product name “JCA-BM 8”, manufactured byJapan Electron Optics Laboratory Co. Ltd.).

[0074] The results are shown in Tables 3 and 4 below and FIGS. 1 and 2.Table 3 and FIG. 1 show the result of the absorbance measurement in thecase where the enzyme reagent samples containing the tetrazoliumcompound were stored at 4° C., and Table 4 and FIG. 2 show the result ofthe absorbance measurement in the case where the enzyme reagent samplescontaining the tetrazolium compound were stored at 25° C.

[0075] (Preparation of Measurement Sample)

[0076] The measurement samples were prepared so as to have the followingcompositions. The hemolysate sample shown below was prepared by freezingblood, storing it, and then melting it to hemolyze blood cells.Hemolysate sample (Hb concentration 100 g/L)  50 μL, 150 μL, 250 μL 20wt % polyoxy lauryl ether  84 μL 1 mol/L glycinamide buffer (pH 9.0)  81μL water remaining portion total amount 750 μL

[0077] (Composition of Color-Developing Reagent) FAOD  26.0 KU/LPeroxidase (POD)  78.0 KU/L Color-developing substrate 0.052 mmol/LPhosphate buffer (pH 6.9)  0.20 mmol/L

[0078] As the FAOD, a product named “Fructosyl Amino Acid Oxidase”(ARKRAY, INC.) was used. Furthermore, as the color-developing substrate,a product named “DA-64” (Wako Pure Chemical Industries, Ltd.) was used.TABLE 3 Storage temperature: 4° C. Absorbance Storage period Ex. 2 Ex. 3Com. Ex. 2  0 day 0.01159 0.00994 0.01652  5 days 0.01797 0.013150.00480  9 days 0.03295 0.02392 0.00967 20 days 0.03591 0.02350 0.0005826 days 0.03502 0.02263 −0.0081 33 days 0.01934 0.01338 −0.0033

[0079] TABLE 4 Storage temperature: 25° C. Absorbance Storage period Ex.2 Ex. 3 Com. Ex. 2  0 day 0.01159 0.00994 0.01652  5 days 0.018880.01335 0.00096  9 days 0.04028 0.02840 0.01206 20 days 0.03220 0.021720.00298 26 days 0.02792 0.02076 −0.0051 33 days 0.01439 0.01125 −0.0048

[0080] With regard to spontaneous color development of the tetrazoliumcompound in the case where the tetrazolium compound was stored as asolution, the color development was suppressed in Examples 2 and 3 farmore strongly than in Comparative Example 2, as can be seen from Table2. Furthermore, with regard to the glycated hemoglobin measurement, inComparative Example 2, the absorbance decreased with time, which meansthat the tetrazolium compound gradually lost its function of eliminatingthe influence of the reducing substance. In contrast, in Examples 2 and3, the absorbance after 30-day storage was substantially the same asthat after 0-day storage, which means that the function of thetetrazolium compound was maintained stably. Still further, with regardto storage conditions, it was found that the tetrazolium compoundexhibits higher stability at pH 6.5, which is close to neutral, than atpH 5.5. Furthermore, as can be seen from FIGS. 1 and 2, in Examples 2and 3, an increase in absorbance was observed, and particularly highabsorbance was maintained 10 to 26 days after the start of the storage.From this fact, it can be said that, when a tetrazolium compound isstored as a solution in the presence of sodium azide, the tetrazoliumcompound can be stabilized and besides, the sensitivity of themeasurement can be improved.

Example 4 and Comparative Example 3

[0081] In Example 4, a tetrazolium compound was stored as an aqueoussolution having a predetermined pH in the presence of sodium azide, andthe change in absorbance of the aqueous solution was examined.

[0082] Samples (A1 to A3, B1 to B3) were prepared so as to have thefollowing compositions and stored at 40° C. The absorbance of thesesamples at the wavelength of 450 nm was measured 3 days and 8 days afterthe start of storage using the above-described spectrophotometer. Thechange in absorbance in each sample over 5 days was determined. A1 A2 A3B1 B2 B3 Type of buffer MES MOPS PIPES MES MOPS PIPES solution pH ofbuffer 5.5 6.5 7.5 5.5 6.5 7.5 solution WST-3 (mmol/L) 0.5 0.5 0.5 2.02.0 2.0 NaN₃ (g/L) 0.05 0.05 0.05 0.1 0.1 0.1

[0083] Furthermore, in Comparative Example 3, samples were prepared inthe same manner as that in Example 4 except that no sodium azide wasadded thereto. The thus-obtained samples (a1 to a3, b1 to b3) ofComparative Example 3 correspond to the samples (A1 to A3, B1 to B3) ofExample 4, respectively. The samples a1 to a3 and b1 to b3 were storedunder the same conditions as those in Example 4, and the change inabsorbance in each sample was determined. The results are shown in Table5 below and FIG. 3A and 3B. FIG. 3A shows the result with respect to thesamples A1 to A3 and a1 to a3, and FIG. 3B shows the result with respectto the samples B1 to B3 and b1 to b3. TABLE 5 Change in absorbance (5days) Ex. 4 Com. Ex. 3 A1 0.036 a1 0.093 A2 0.083 a2 0.105 A3 0.133 a30.258 B1 0.033 b1 0.058 B2 0.023 b2 0.161 B3 0.430 b3 0.663

[0084] As can be seen from Table 5, in the samples (A1 to A3 and B1 toB3) of Example 4, the change in absorbance was less than that in thecorresponding samples (a1 to a3 and b1 to b3) of Comparative Example 3.Therefore, it can be said that a tetrazolium compound can be storedstably in the presence of sodium azide.

[0085] Industrial Applicability

[0086] As specifically described above, according to a method of thepresent invention, a tetrazolium compound can be stored stably not onlyunder acidic conditions but also under other pH conditions. Therefore,when a liquid reagent containing a tetrazolium compound is needed, it isnot necessary to prepare a reagent for each use. This allows the reagentto be produced at low cost and also simplifies the operation.

1. A method for storing a tetrazolium compound stably, comprising:storing the tetrazolium compound in the presence of sodium azide.
 2. Themethod according to claim 1, wherein the tetrazolium compound (A) andthe sodium azide (B) are present at a ratio (molar ratio A:B) in a rangefrom 1:0.02 to 1:6.2.
 3. The method according to claim 1 or 2, whereinthe tetrazolium compound and the sodium azide are present in a solution,and a concentration of the tetrazolium compound is in a range from 0.5to 8 mmol/L.
 4. The method according to any one of claims 1 to 3,wherein the tetrazolium compound and the sodium azide are present in asolution, and a concentration of the sodium azide is in a range from0.08 to 3.2 mmol/L.
 5. The method according to claim 3 or 4, wherein apH of the solution is in a range from 5.0 to 7.5.
 6. The methodaccording to any one of claims 1 to 5, wherein the tetrazolium compoundis2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliumsalt.
 7. The method according to any one of claims 1 to 6, wherein astorage temperature is in a range from 4° C. to 60° C.
 8. A stabilizerfor storing a tetrazolium compound stably, comprising sodium azide.
 9. Areagent solution comprising an aqueous solvent and a tetrazoliumcompound dissolved in the aqueous solvent, wherein sodium azide furtheris dissolved in the aqueous solvent.
 10. A dry reagent containing atetrazolium compound, obtained by drying an aqueous solvent in which thetetrazolium compound and sodium azide are dissolved.